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Title:  Compositions and methods for identifying antigens which elicit an immune response

United States Patent:  6,716,623

Issued:  April 6, 2004

Inventors:  Chen; Si-Yi (Pearland, TX); You; Zhaoyang (Houston, TX)

Assignee:  Wake Forest University School of Medicine (Winston-Salem, NC)

Appl. No.:  201764

Filed:  July 22, 2002

Abstract

This invention relates to an expression vector wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen protein or peptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked. More particularly, it relates to the method of eliciting an immune response directed against an antigen in a mammal comprising the steps of introducing the expression vector into a cell, expressing the vector to produce an antigen under conditions wherein the antigen is secreted from the cell, endocytosing the secreted antigen into the cell, processing the antigen, and presenting fragments to a receptor to elicit a T-cell response. In addition, this invention relates to a vaccine and a method of use. The invention also relates to the method of identifying MHC-II restricted epitopes.

SUMMARY OF THE INVENTION

An embodiment of the present invention is an expression vector comprising a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked.

In specific embodiments of the present invention, the polynucleotide promoter sequence is selected from the group consisting of a constitutive promoter, an inducible promoter and a tissue specific promoter.

In another specific embodiment of the present invention, the polynucleotide encoding a signal sequence is selected from the group consisting of a hepatitis B virus E antigen signal sequence, an immunoglobulin heavy chain leader sequence, and a cytokine leader sequence.

An embodiment of the present invention is an expression vector wherein the polynucleotide encoding an antigen comprises a polynucleotide sequence for at least one epitope, wherein said at least one epitope induces a B cell response in a mammal.

A further embodiment of the present invention is an expression vector wherein the polynucleotide encoding an antigen comprises a polynucleotide sequence for at least one epitope, wherein said at least one epitope induces a CD4+ T-cell response in a mammal.

Another embodiment of the present invention is an expression vector wherein the polynucleotide encoding an antigen comprises a polynucleotide sequence for at least one epitope, wherein said at least one epitope induces a CD8+ T-cell response in a mammal.

A specific embodiment of the present invention is an expression vector wherein the polynucleotide sequence encoding an antigen comprises a polynucleotide sequence for at least one epitope, wherein said at least one epitope induces a B cell response, a CD4+ T-cell response and a CD8+ T-cell response in a mammal into which said antigen is introduced.

A further specific embodiment of the present invention is an expression vector wherein the polynucleotide sequence encoding an antigen comprises a polynucleotide sequence for a plurality of epitopes, wherein said plurality of epitopes induces a B cell response, a CD4+ T-cell response and a CD8+ T-cell response in a mammal into which said antigen is introduced.

A further embodiment of the present invention is an expression vector wherein the polynucleotide encoding a cell binding element is a polynucleotide sequence of a ligand which binds to a cell surface receptor. In specific embodiments, the cell binding element sequence is selected from the group consisting of polynucleotide sequences which encode a Fc fragment, a toxin cell binding domain, a cytokine, a small peptide and an antibody. In specific embodiments, the polynucleotide encoding a cell binding element is a homologous polynucleotide sequence or a heterologous polynucleotide sequence.

An additional embodiment of the present invention is a transformed cell comprising an expression vector wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked.

Another specific embodiment of the present invention is a fusion protein wherein the fusion protein comprises a signal sequence, an antigen and a cell binding element. In specific embodiments, antigen presenting cells have been transduced with the fusion protein in vitro. (In further embodiments, the fusion protein is administered directly to a mammal.

A specific embodiment of the present invention is a vaccine comprising an expression vector wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked. In specific embodiments, a vaccine comprises antigen presenting cells, wherein said antigen presenting cells are transduced in vitro with the expression vector. In further embodiments, a vaccine comprises antigen presenting cells, wherein said antigen presenting cells are transduced in vitro with the fusion protein.

Another specific embodiment of the present invention is an expression vector comprising at least a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen and a polynucleotide encoding a cell binding element.

A further embodiment of the present invention is a method to elicit an immune response directed against an antigen, comprising the steps of: introducing an expression vector into a cell, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked; and expressing said vector to produce an antigen under conditions wherein said antigen is secreted from the cell; said secreted antigen is endocytosed into the cell; said endocytosed antigen is processed inside the cell; and said processed antigen is presented to a cell surface protein, to elicit a T-cell mediated immune response. In specific embodiments, the antigen is secreted by a first cell and internalized by a second cell wherein the first and second cells are antigen presenting cells. In further embodiments, the first cells is a non-antigen presenting cell and the second cell is an antigen presenting cell.

Another specific embodiment of the present invention is a method to identify a polynucleotide sequence which encodes at least one MHC-II restricted epitope that is capable of activating CD4+ helper T-cells, said method comprising the steps of: introducing an expression vector into an antigen presenting cell to produce a transduced antigen presenting cell, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked; contacting said transduced antigen presenting cell with naive or primed T-cells; and assessing whether any naive T-cells or primed T-cells are activated upon contact with said transduced antigen presenting cell, wherein activation of any of said T-cells indicates that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells. In specific embodiments, the polynucleotide encoding a test polypeptide is selected from the group of cDNA libraries consisting of viral genomes, bacterial genomes, parasitic genomes and human genomes.

Another embodiment of the present invention is a method to identify a polynucleotide sequence which encodes at least one MHC-II restricted epitope that is capable of eliciting an immune response in vivo, said method comprising the steps of: introducing an expression vector into antigen presenting cells to produce transduced antigen presenting cells, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked; administering said transduced antigen presenting cells to a mammal via a parenteral route; collecting T-cells from splenocytes and co-culturing with dendritic cells; and assessing activation of T-cells, wherein said activation of T-cells indicate that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells. In specific embodiments, the polynucleotide encoding a test polypeptide is selected from the group of cDNA libraries consisting of viral genomes, bacterial genomes, parasitic genomes and human genomes.

A specific embodiment of the present invention is a method to identify a polynucleotide sequence which encodes at least one MHC-II restricted epitope that is capable of eliciting an immune response in vivo, said method comprising the steps of: administering to a mammal via parenteral route an expression vector, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked; administering said transduced antigen presenting cells to a mammal via a parenteral route; collecting T-cells from splenocytes and co-culturing with dendritic cells; and assessing activation of T-cells, wherein said activation of T-cells indicate that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells. In specific embodiments, the polynucleotide encoding a test polypeptide is selected from the group of cDNA libraries consisting of viral genomes, bacterial genomes, parasitic genomes and human genomes.

A specific embodiment of the present invention is a method of treating cancer comprising the steps of identifying a test polypeptide which encodes at least one MHC-II restricted epitope, wherein said polypeptide is identified under the conditions of transducing antigen presenting cells with an expression vector into antigen presenting cells to produce transduced antigen presenting cells, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and assessing activation of T-cells, wherein said activation of T-cells indicate that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells; and administering antigen presenting cells to a mammal via a parenteral route, wherein said antigen presenting cells are transduced with the test polypeptide.

Another specific embodiment of the present invention is a method of treating cancer comprising the steps of identifying a test polypeptide which encodes at least one MHC-II restricted epitope, wherein said polypeptide is identified under the conditions of transducing antigen presenting cells with an expression vector into antigen presenting cells to produce transduced antigen presenting cells, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and assessing activation of T-cells, wherein said activation of T-cells indicate that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells; and administering to a mammal via a parenteral route an expression vector, wherein said expression vector comprises at least the polynucleotide encoding the test polypeptide and a polynucleotide encoding a cell binding element said antigen presenting cells are transduced with the test polypeptide.

A further specific embodiment of the present invention is a method of treating a viral infection comprising the steps of identifying a test polypeptide which encodes at least one MHC-II restricted epitope, wherein said polypeptide is identified under the conditions of transducing antigen presenting cells with an expression vector into antigen presenting cells to produce transduced antigen presenting cells, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and assessing activation of T-cells, wherein said activation of T-cells indicate that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells; and administering antigen presenting cells to a mammal via a parenteral route, wherein said antigen presenting cells are transduced with the test polypeptide.

Another embodiment of the present invention is a method of treating a viral infection comprising the steps of identifying a test polypeptide which encodes at least one MHC-II restricted epitope, wherein said polypeptide is identified under the conditions of transducing antigen presenting cells with an expression vector into antigen presenting cells to produce transduced antigen presenting cells, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and assessing activation of T-cells, wherein said activation of T-cells indicate that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells; and administering to a mammal via a parenteral route an expression vector, wherein said expression vector comprises at least the polynucleotide encoding the test polypeptide and a polynucleotide encoding a cell binding element said antigen presenting cells are transduced with the test polypeptide.

Another embodiment of the present invention is a method of treating, an autoimmune disease comprising the steps of identifying a test polypeptide which encodes at least one MHC-II restricted epitope, wherein said polypeptide is identified under the conditions of transducing antigen presenting cells with an expression vector into antigen presenting cells to produce transduced antigen presenting cells, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and assessing activation of T-cells, wherein said activation of T-cells indicate that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells; and administering antigen presenting cells to a mammal via a parenteral route, wherein said antigen presenting cells are transduced with the test polypeptide.

A specific embodiment of the present invention is a method of treating an autoimmune disease comprising the steps of identifying a test polypeptide which encodes at least one MHC-II restricted epitope, wherein said polypeptide is identified under the conditions of transducing antigen presenting cells with an expression vector into antigen presenting cells to produce transduced antigen presenting cells, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a test polypeptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and assessing activation of T-cells, wherein said activation of T-cells indicate that the polynucleotide encoding the test polypeptide is a gene or fragment thereof capable of activating CD4+ helper T-cells; and administering to a mammal via a parenteral route an expression vector, wherein said expression vector comprises at least the polynucleotide encoding the test polypeptide and a polynucleotide encoding a cell binding element said antigen presenting cells are transduced with the test polypeptide.

A further embodiment of the present invention is a method of producing a vaccine to immunize a mammal comprising the steps of: transducing antigen presenting cell by introducing an expression vector into a cell, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked; and expressing said vector to produce an antigen under conditions wherein said antigen is secreted from the cell. In specific embodiments, antigen presenting cells are transduced with the antigen in vitro or ex vivo prior to administering the antigen presenting cells to the mammal.

Another specific embodiment of the present invention is a method of inducing an immune response comprising the steps of co-administering to a mammal a cytokine expression vector and a retrogen expression vector, wherein the retrogen expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked.

A further embodiment of the present invention is a method of inducing an immune response comprising the steps of co-administering to a mammal one expression vector, wherein said expression vector comprises a polynucleotide sequence encoding a cytokine protein and a polynucleotide sequence encoding a fusion protein under transcriptional control of one promoter, wherein said fusion protein comprises an antigen and a cell binding element. In specific embodiments, the polynucleotide sequence encoding the cytokine protein and the polynucleotide sequence encoding the fusion protein are under separate transcriptional control, and wherein the polynucleotide sequence encoding the cytokine protein and the polynucleotide sequence encoding the fusion protein are in tandem in the one expression vector.

Another embodiment of the present invention is a method of inducing an immune response comprising the steps of co-administering to a mammal two different retrogen expression vectors, wherein a first retrogen expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a first antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked; and a second retrogen expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a second antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively.

Another specific embodiment of the present invention is a method of inducing an immune response comprising the steps of administering to a mammal one expression vector, wherein said expression vector comprises a polynucleotide sequence encoding a first fusion protein and a polynucleotide sequence encoding a second fusion protein under transcriptional control of one promoter, wherein said first fusion protein comprises a first antigen and a first cell binding element and said second fusion protein comprises a second antigen and a first cell binding element. In specific embodiments, the first and second antigens are different antigens and the cell binding elements is a Fc fragment. In further embodiments, the first and second antigens are different antigens and the first and second cell binding elements are different cell binding elements. An additional embodiment includes that the polynucleotide sequence encoding the first fusion protein and the polynucleotide sequence encoding the second fusion protein are under separate transcriptional control, and wherein the polynucleotide sequence encoding the first fusion protein and the polynucleotide sequence encoding the second fusion protein are in tandem in one expression vector.

A specific embodiment of the present invention is a method of simultaneously inducing both CD4+ and CD8+ T-cells comprising the steps of administering a fusion protein wherein the protein comprises both a MHC-I and MHC-II epitope fused to a cell binding element.

A further embodiment of the present invention is a method of producing a fusion protein comprising the steps of introducing an expression vector into a cell, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and expressing said vector to produce a fusion protein under conditions wherein said fusion protein is secreted from the cell. In specific embodiments, antigen presenting cells are transduced with the fusion protein in vitro.

A specific embodiment of the present invention is a method of secreting an intracellular protein comprising the steps of introducing an expression vector into a cell, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an intracellular protein, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and expressing said vector to produce a fusion protein under conditions wherein said fusion protein is secreted from the cell. More specifically, the polynucleotide sequence encoding the intracellular protein is truncated or mutated to increase efficiency of secretion.

Another specific embodiment of the present invention is a method of secreting a membrane protein comprising the steps of introducing an expression vector into a cell, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a membrane protein, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and expressing said vector to produce a fusion protein under conditions wherein said fusion protein is secreted from the cell. More specifically, the polynucleotide sequence encoding the membrane protein is truncated or mutated to increase efficiency of secretion.

DETAILED DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that various embodiments and modifications may be made to the invention disclosed in this Application without departing from the scope and spirit of the invention.

One embodiment of the present invention is an expression vector comprising a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked.

In specific embodiments, the nucleic acid sequence encoding a fusion protein (antigen-cell binding element) is under transcriptional control of a promoter. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements, i.e., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR.TM., in connection with the compositions disclosed herein (U.S. Pat. Nos. 4,683,202, 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

A promoter sequence exemplified in the experimental examples presented herein is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HW) long terminal repeat (LTR) promoter, Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the muscle creatine promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Further, the invention includes the use of a tissue specific promoter, which promoter is active only in a desired tissue. Tissue specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and the PSA associated promoter sequences.

In specific embodiments of the present invention, the expression vector comprises a polynucleotide encoding a signal sequence, which directs processing of the protein encoded thereby to the appropriate cellular machinery in order that the protein is secreted from the cell. Exemplary signal sequences include, but are not limited to, hepatitis B virus E antigen signal sequence, immunoglobulin heavy chain leader sequences, cytokine leader sequences, and the like, can be used. Essentially, any signal sequence that directs secretion of a protein from a cell is suitable for use in the expression vector of the invention. In addition to signal sequences, other mechanisms for secretion may be employed, such as but not limited to, truncation or deletion of sequences inhibiting protein secretion, point mutations of sequences inhibiting protein secretion, and linkage of the protein to a viral gene to be assembled into viral particles.

An embodiment of the present invention is an expression vector wherein the polynucleotide encoding an antigen comprises a polynucleotide sequence for at least one epitope, wherein said at least one epitope induces a B cell response in a mammal.

A further embodiment of the present invention is an expression vector wherein the polynucleotide encoding an antigen comprises a polynucleotide sequence for at least one epitope, wherein said at least one epitope induces a CD4+ T-cell response in a mammal.

Another embodiment of the present invention is an expression vector wherein the polynucleotide encoding an antigen comprises a polynucleotide sequence for at least one epitope, wherein said at least one epitope induces a CD8+ T-cell response in a mammal.

A specific embodiment of the present invention is an expression vector wherein the polynucleotide sequence encoding an antigen comprises a polynucleotide sequence for at least one epitope, wherein said at least one epitope induces a B cell response, a CD4+ T-cell response and a CD8+ T-cell response in a mammal into which said antigen is introduced.

A further specific embodiment of the present invention is an expression vector wherein the polynucleotide sequence encoding an antigen comprises a polynucleotide sequence for a plurality of epitopes, wherein said plurality of epitopes induces a B cell response, a CD4+ T-cell response and a CD8+ T-cell response in a mammal into which said antigen is introduced.

In specific embodiments of the present invention, the expression vector comprises a polynucleotide sequence encoding an antigen. The polynucleotide sequences encoding an antigen are selected from at least one polynucleotide sequence associated with a disease, wherein said disease is selected from the group consisting of infectious disease, cancer and autoimmune disease. More particularly, the polynucleotide sequence encoding the antigen is a polynucleotide sequence selected from the group of pathogenic microorganisms that cause infectious disease consisting of virus, bacterium, fungus and protozoan. These DNA sequences encoding known proteins or fragments thereof include viral antigens, such as but not limited to, hepatitis B and hepatitis C virus antigens, human immunodeficiency virus antigens, including but not limited to, gp160, gp120 and gag proteins, papillomavirus antigens, including but not limited to the E7 and E6 proteins. Herpes virus proteins, such as for example, proteins encoded by Epstein-Barr virus, cytomegalovirus, herpes simplex virus types 1 and 2, and human herpes viruses 6, 7 and 8, are also contemplated in the invention as useful retrogen fusion proteins. In further embodiments, the polynucleotide encoding an antigen is a polynucleotide selected from the group consisting of breast cancer, cervical cancer, melanoma, renal cancer and prostate cancer. In addition, the protein can be one, which induces activation of an immune response directed against tumor cells for the purpose of inhibiting their growth and replication, i.e., tyrosinase that activates an immune response against melanocytes in melanoma. Other tumor-associated proteins include, but are not limited to, MART, trp, MAGE-1, MAGE-2, MAGE-3, gp100, HER-2, PSA, the Ras antigen associated with lung cancer and any other tumor specific, tissue specific or tumor associated antigens. One skilled in the art is aware of known polynucleotide sequences, which encode tumor associated antigens, as well as, are well documented in the scientific literature and heretofore unknown polynucleotide sequences are being discovered with great rapidity. In a further embodiment, the polynucleotide sequence encoding an antigen is selected from an autoimmune disease including, but not limited to, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis and Crohn's disease. In addition, the invention should be construed to include DNA that encodes an autoantigen in order to induce immune tolerance in situations in which such tolerance is of benefit to the mammal. Further, the invention should be construed to include DNA that encodes an antigen, which is capable of inducing a generalized immune response in a mammal where a generalized immune response is of benefit to the mammal. A generalized immune response may be of benefit to the mammal in instances wherein the mammal is immunosuppressed, i.e., as a result of HIV infection, chemotherapy, or other immunosuppressive procedures. Such antigens may include, but are not limited to, Fc antibody fragments which when bound to Fc receptors on antigen presenting cells serve to upregulate antigen presentation by these cells. In addition, interleukins, such as, but not limited to, interleukin 5 may be used to generate a similar adjuvant effect in mammals in which induction of a generalized immune response is desired. The present invention should therefore be construed to include any known or heretofore unknown polynucleotide sequences which when included in the expression vector of the invention are capable of activating the immune response when the vector, or the fusion protein encoded thereby, is introduced into a mammal.

One skilled in the art is cognizant that it is not necessary that the nucleic acid sequence encode a full-length protein. It is simply necessary that the expressed protein comprise an epitope, which elicits the desired immune response when processed in antigen presenting cells. Thus, it is apparent from this information that the nucleic acid sequence may encode any antigen which can elicit an immune response in the animal into which the expression vector is introduced. Thus, the invention should in no way be limited to the type of nucleic acid sequences contained within the expression vector, but should include any and all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, including also without limitation, the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR.TM., and the like, and by synthetic means. The invention also should not be construed to be limited in any way to the source of the nucleic acid sequence, in that nucleic acid sequence may be obtained from any available source. One skilled in the art is aware that protocols for obtaining a nucleic acid sequence are well known in the art and are described, for example in Sambrook et al. (1989), and in Ausubel et al. (1997).

In specific embodiments of the present invention, the expression vector further comprises a polynucleotide sequence encoding a cell binding element. The cell binding element is a portion of a polypeptide, which facilitates binding of a protein to a cell receptor. A polynucleotide encoding any ligand that binds to a cell receptor protein may be used in the expression vector of the invention. Exemplary cell binding elements include, but are not limited to, immunoglobulin Fc fragment, toxin receptor protein cell binding domains, such as for example, the pseudomonas exotoxin cell binding domain, a cytokine, for example, interleukin 5, and interleukin 6, any type of an antibody molecule, and the like. A skilled artisan is cognizant that any antibody is capable of binding to cell surface markers on the surface of antigen presenting cells initiating internalization of the antigen/antibody complex. Thus, an antibody or a fragment thereof can be used as a cell binding element to initiate internalization. Exemplary antibodies include, but should not be limited to, antiCDC11, antiCD54, antiCD80, and antiCD86. Furthermore, one skilled in the art is cognizant that the cell binding element can be homologous or heterologous. For example, the Fc fragment can be homologous or heterologous. Thus, the invention should not be construed to be limited in any way to the source of the cell binding element, in that the sequence for a cell binding element may be obtained from any available source including, without limitation, the cloning of DNA from a recombinant library or a cell genome, using ordinary cloning technology and PCR.TM., and the like, and by synthetic means.

In addition to using portions of known binding elements, a skilled artisan is cognizant that small peptides could be identified via a typical screening procedure well known in the art. A DNA library (cDNA or genomic) is screened to identify small peptides that bind efficiently to antigen presenting cells. Once these peptides are identified, the peptide is sequenced and used as a cell binding element in the present invention.

In expression, one will typically include a polyadenylation sequence to effect proper polyadenylation of the transcript. The nature of the polyadenylation sequence is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation sequence, LTR polyadenylation sequence, and/or the bovine growth hormone polyadenylation sequence, convenient and/or known to function well in various target cells. Also contemplated as an element of the expression vector is a transcriptional termination site. These elements can serve to enhance message levels and/or to minimize read through from the inserted polynucleotide sequences encoding the antigen and cell binding elements into other sequences of the vector.

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast. In instances wherein it is beneficial that the expression vector replicate in a cell, the vector may integrate into the genome of the cell by way of integration sequences, i.e., retrovirus long terminal repeat sequences (LTRs), the adeno-associated virus ITR sequences, which are present in the vector, or alternatively, the vector may itself comprise an origin of DNA replication and other sequence which facilitate replication of the vector in the cell while the vector maintains an episomal form. For example, the expression vector may optionally comprise an Epstein-Barr virus (EBV) origin of DNA replication and sequences which encode the EBV EBNA-1 protein in order that episomal replication of the vector is facilitated in a cell into which the vector is introduced. For example, DNA constructs having the EBV origin and the nuclear antigen EBNA-1 coding are capable of replication to high copy number in mammalian cells and are commercially available from, for example, Invitrogen (San Diego, Calif.).

It is important to note that in the present invention it is not necessary for the expression vector to be integrated into the genome of the host cell for proper protein expression. Rather, the expression vector may also be present in a desired cell in the form of an episomal molecule. For example, there are certain cell types in which it is not necessary that the expression vector replicate in order to express the desired protein. These cells are those which do not normally replicate, such as muscle cells, and yet are fully capable of gene expression. An expression vector may be introduced into non-dividing cells and express the protein encoded thereby in the absence of replication of the expression vector.

To identify cells that contain the nucleic acid constructs of the present invention, the cells are identified in vitro or in vivo by including a marker in the expression vector. Such markers confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one, in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers in conjunction with FACS analysis. For example, NGFR (nerve growth factor receptor) is included in the expression vector to facilitate selection of cells comprising the vector by using a flow cytometric assay detecting NGFR expression on the cell surface. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

The expression vector may also comprise a prokaryotic origin of DNA replication and a gene encoding a detectable marker for selection of prokaryotic cells comprising the expression vector, for example, an antibiotic resistance gene, such as, for example, the ampicillin resistance gene.

In addition, the expression vector may be provided to the cell in the form of RNA instead of DNA. The core components of the vector are the same as those described herein for a DNA vector, and in addition, other components may be added which serve to stabilize the RNA in bodily fluids and in tissues and cells.

The actual methods of ligating together the various components described herein to generate the expression vector of the invention are well known in the art and are described, for example, in Sambrook et al. (1989), Ausubel et al. (1994), and in Gerhardt et al. (1994).

In specific embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors and mammalian vectors. Numerous expression vector systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986, 4,879,236 and can be bought, for example, under the name MAXBAC.RTM. 2.0 from INVITROGEN.RTM. and BACPACK.TM. BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH.RTM..

Other examples of expression vector systems include STRATAGENE.RTM.'S COMPLETE CONTROL.TM. Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN.RTM., which carries the T-REX.TM. (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN.RTM. also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

A transformed cell comprising an expression vector is generated by introducing into the cell the expression vector. The introduction of DNA into a cell or host cell is well known technology in the field of molecular biology and is described, for example, in Sambrook et al. (1989), Ausubel et al. (1994), and in Gerhardt et al., (1994). Methods of transfection of cells include calcium phosphate precipitation, liposome mediated transfection, DEAE dextran mediated transfection, electroporation and the like. Alternatively, cells may be simply transduced with the retrogen expression vector of the invention using ordinary technology described in the references and examples provided herein. The host cell includes a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). It is well within the knowledge and skill of a skilled artisan to determine an appropriate host. Generally this is based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5.alpha., JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE.RTM. Competent Cells and SOLOPACK.TM. Gold Cells (STRATAGENE.RTM., La Jolla, Calif.). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to yeast, insects and mammals. Examples of mammalian eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Examples of yeast strains include, but are not limited to, YPH499, YPH500 and YPH501. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either an eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (1989), and in Ausubel et al. (1994), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

A specific embodiment of the present invention is a fusion protein comprising a signal sequence an antigen and a cell binding element. The invention also includes the use of the retrogen protein or fusion protein as a vaccine. The retrogen protein may be obtained by expressing the retrogen protein in any cell comprising the expression vector and separating the retrogen protein from the cell, cell debris and cell medium. Affinity column purification procedures may be especially useful for purification of the retrogen of the invention because the retrogen, by definition comprises a cell binding element. An affinity column comprising the matching cellular receptor, or a generic protein such as protein A or protein G, may be used to separate the retrogen from the cellular components. Another embodiment is a vaccine comprising antigen presenting cells that are transduced in vitro with the fusion protein.

In further embodiments, a vaccine comprises the expression vector, wherein said expression vector comprises a polynucleotide sequence encoding a promoter sequence, a polynucleotide sequence encoding a secretion signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide encoding a polyadenylation sequence, all operatively linked. The vaccine comprising the expression vector is administered directly to the mammal to sites in which there are cells into which the sequences contained within the vector may be introduced, expressed and an immune response against the desired protein may be elicited. In this instance, the expression vector is administered in a pharmaceutical carrier and in a formulation such that the DNA is capable of entering cells, and being expressed therein. The expressed protein may then enter antigen presenting cells for processing and MHC presentation as described herein. A skilled artisan realizes that the DNA may be given in a variety of ways and, depending upon the route of injection, the composition of DNA may need to be manipulated. Exemplary routes of parenteral injections include, but are not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous and intradermal. Further, it is not necessary that the DNA of the expression vector be introduced into the cells of the mammal by direct injection of the same into the tissues of the mammal. Rather, other means of introduction of the expression vector into the mammal may be used, including, but not limited to non-invasive pressure injection, nasal, oral, etc.

The amount of DNA which is to be introduced into the mammal is an amount sufficient for efficient expression of the DNA in the cell, such that a sufficient amount of protein is expressed and secreted therefrom, which protein is then taken up by antigen presenting cells and expressed thereon as an MHC complex. Such an amount of DNA is referred to herein as a "therapeutic amount" of DNA. The precise concentration of DNA which constitutes a therapeutic amount may be easily determined by one skilled in the art of administration of such compounds to mammals, and will of course vary depending on the components contained therein, and other factors including, but not limited to, the tissue into which the DNA is being introduced and the age and health of the mammal.

Another specific embodiment of the present invention is a vaccine comprising cells that are transduced with the expression vector. These transduced cells are in the form of a pharmaceutical composition for administration to a mammal for the purpose of eliciting an immune response therein. Expression of the retrogen protein in the cells results in secretion of the retrogen protein from the cells. Secreted retrogen protein may then be taken up by antigen presenting cells in the mammal for processing therein and expression therefrom as a MHC-I or a MHC-II complex. When the eukaryotic cell is an antigen presenting cell, the retrogen protein may be expressed therein, secreted therefrom and may reenter the cell for processing and antigenic MHC presentation. When the eukaryotic cell is not an antigen presenting cell, the cell expresses and secretes the retrogen protein, which is subsequently taken up by an antigen presenting cell for antigenic MHC presentation. Non-antigen presenting cells useful in the invention include any cell which does not process antigens for MHC presentation, i.e., muscle cells. Antigen presenting cells include dendritic cells (DC), macrophages, monocytes and the like. Tumor cells, which are also included, may be cells, which are or are not capable of processing antigens for MHC presentation.

The expression vector may also be introduced into stem cells of a mammal, either directly in vivo in the mammal, or more preferably, ex vivo in cells which are removed from the mammal and are reintroduced into the mammal following introduction of the vector into the cells. The expression vector may also be introduced into other cells in the mammal in an ex vivo approach. When the vector is introduced into cells in the mammal, it is not necessary that the vector express the protein encoded thereby immediately, in that, it may be more desirable that the protein be expressed in the cells at some later time. In this instance, the expression vector preferably comprises an inducible promoter, which is activated upon administration of the appropriate inducer to the mammal or to cells of the mammal. Ex vivo technology is well known in the art and is described, for example, in U.S. Pat. No. 5,399,346.

A further embodiment is an expression vector comprising at least a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen and a polynucleotide encoding a cell binding domain.

Another embodiment of the present invention is a method to elicit an immune response directed against an antigen. More particularly, this method utilizes the expression vector of the present invention to manipulate cells to produce endogenous antigens as if they were exogenous antigens. This novel antigen presentation strategy involves transducing cells with a novel recombinant expression vector to produce and secrete a fusion-protein consisting of an antigen and a cell-binding element. The secreted fusion protein is endocytosed or "retrogradely" transported into antigen presenting cells via receptor-mediated endocytosis (Daeron, 1997; Serre et al., 1998; Ravetch et al., 1993). As a result, the fusion protein, or "retrogen" as termed in the present disclosure because of its retrograde transport following secretion, is processed in the endosomal pathway and is presented on the cell surface of the antigen presenting cells as an MHC-II restricted exogenous antigenic fragments even though it has been produced endogenously. The MHC-II bound antigenic fragments of the antigen on the surface of the antigen presenting cells activate CD4+ T-cells that in turn stimulate CD8+ T-cells and macrophages, as well as B-cells to induce both cellular and humoral immunity.

It has also been discovered in the present invention that the retrogen protein may also be processed in the cytosolic pathway during the fusion protein synthesis, secretion and endocytosis and become associated with MHC-I on the surface of the antigen presenting cells to directly activate CD8+ T-cells. Activation of CD8+ T cells by internalized antigens is described in the art and for example, in Kovacsovics-Bankowski et al., 1995. In addition, as noted above and described in more detail elsewhere herein, B cells may be activated by the secreted retrogen. Thus, B cell activation is enhanced markedly in the present system in that CD4+ cells also activates B cells. Thus, this strategy uses a unifying mechanism to activate all of the arms of the immune system.

In specific embodiments, the expression vector is introduced into a cell to produce a transduced cell. Expression of the retrogen protein in the cells results in secretion of the retrogen protein from the cells. Secreted retrogen protein may then be taken up by antigen presenting cells in the mammal for processing therein and expression therefrom as a MHC-I or a MHC-II complex. Thus, one skilled in the art realizes that the transduced cell or first cell, secretes the antigen and the secreted antigen is internalized into a cell, a second cell, either the same cell or a different cell. When the eukaryotic cell is an antigen presenting cell, the retrogen protein may be expressed therein, secreted therefrom and may reenter the cell for processing and antigenic MHC presentation. When the eukaryotic cell is not an antigen presenting cell, the cell expresses and secretes the retrogen protein, which is subsequently taken up by an antigen presenting cell for antigenic MHC presentation. Non-antigen presenting cells useful in the invention include any cell which does not process antigens for MHC presentation, i.e., muscle cells. Antigen presenting cells include dendritic cells (DC), macrophages, monocytes and the like. Tumor cells, which are also included, may be cells, which are or are not capable of processing antigens for MHC presentation.

A further embodiment of the present invention, is a method to elicit an immune response directed against an antigen comprising the step of administering the expression vector directly to a mammal.

The invention also includes a method of screening or identifying a polynucleotide sequence which encodes at least one MHC-II restricted epitope that is capable of eliciting an immune response in a mammal. Preferably, the polypeptide, which is identified, is one which elicits an immune response that is beneficial to the mammal. The method comprises obtaining a population of isolated DNA molecules and screening for those isolated DNA molecules which encode at least one MHC-II restricted epitope that is capable of activating CD4+ helper T-cells. The DNA molecules are referred to herein as "test DNA" or "test polynucleotide sequence." The test polynucleotide sequences are cloned into the expression vector of the present invention, in the vector which is positioned between the signal sequence and the cellular binding element as depicted, for example, in FIG. 25. In the method, antigen presenting cells are transduced by introducing the vector comprising the test polynucleotide sequence into the antigen presenting cells, transduced antigen presenting cells are contacted with naive T-cells or primed T-cells and the ability of the transduced cells to activate naive CD4+ T cells in vitro is assessed by assessing whether any naive T-cells or primed T-cells are activated upon contact with said transduced antigen presenting cell. Activation of T cells by transduced antigen presenting cells is an indication that the test polynucleotide sequence contained therein is a polynucleotide sequence, or gene or fragment thereof which encodes at least one epitope capable of activating CD4+ helper T-cells to elicit an immune response in a mammal. Suitable controls which can be used in the assay include cells which are transduced with an expression vector comprising an isolated polynucleotide sequence which is known not to activate the immune response in an mammal (negative control), and cells which are transduced with an expression vector comprising an isolated polynucleotide sequence which is known to activate the immune response in an mammal (positive control). One skilled in the art is cognizant that this screening procedure can be utilized to screen the human genome to identify genes that encode proteins or epitopes that are recognized by CD4+ T-cells that could be used for immunotherapy for cancer or autoimmune disease or for gene therapy. Furthermore, other genomes can be screened including bacterial, viral, or parasitic.

The in vitro T-cell activation assay may be adapted to be a high-throughput automated assay in order to facilitate the testing of many different test polynucleotide sequences at one time. One skilled in the art recognizes that the present invention can be manipulated to transduce cells with expression vectors containing a variety of possible epitope sequences. The transduced cells may be placed in 96-well plates, containing naive T-cells, and the activation of the T-cells may be assessed by automated assessment of incorporation of radioactivity into the DNA of the T-cells, using technology readily available in clinical immunology.

In further embodiments, the protein product encoded by the test polynucleotide sequences may be further evaluated to assess activation of the immune response in a mammal in vivo. This assay is the same as the in vitro assay except, the transduced antigen presenting cells that were transduced by introducing the expression vector comprising the test polynucleotide sequences are administered to a mammal via a parenteral route. In specific embodiments, the expression vector comprising the test polynucleotide sequences is administered directly to a mammal. T-cells are collected from splenocytes and co-cultured with dendritic cells. The activation of T-cells is assessed to determine if the test polynucleotide encoding the test polypeptide is a capable of activating CD4+ helper T cells. Furthermore, one skilled in the art is cognizant that this screening procedure could be utilized to identify MHC-II restricted epitopes that could be use to treat cancer, viral infections and autoimmune disease.

As noted herein, the test polynucleotide sequences may be obtained by any ordinary means common in the art of molecular biology. For example, test polynucleotide sequences may be obtained from an expression library, which library may express proteins whose function is unknown. Test polynucleotide sequences may also be obtained from an expression library which expresses proteins of known function, but which have not heretofore been known to possess the property of activation of the immune system in an mammal. Exemplary expression cDNA libraries include, but are not limited to, tumor cells, viral genomes, bacterial genomes, parasitic genomes, and human genomes. Test polynucleotide sequences may also be obtained using combinatorial methodology, wherein it is not known at the outset whether the polynucleotide sequence encodes a protein, and moreover, it is not known whether the polynucleotide sequence encodes a protein which is capable of activating the immune response. Test polynucleotide sequences may also be obtained by synthetic methods, wherein a polynucleotide sequence is synthesized in an automated synthesizer, fragments of discrete lengths are cloned into the expression vector and are tested as described herein.

It is not always necessary that the immune response be protective, but merely that it be beneficial to the host mammal. For example, it may be beneficial to a mammal to induce immune tolerance in situations wherein an immune response to an antigen is detrimental to the mammal, for example, in certain autoimmune diseases such as rheumatoid arthritis, systemic lupus erythrematosus, psoriasis, multiple sclerosis, Crohn's disease, etc., a diminution in the immune response is desired which can be achieved by inducing immune tolerance against the offensive antigen. In this instance, the DNA comprises DNA encoding the offensive antigen which is then expressed in cells of the mammal and subsequently processed in antigen presenting cells so as to be expressed on the surface thereof as an MHC-I and/or an MHC-II complex in order to induce immune tolerance in the mammal against the antigen.

In a further embodiment, an identified polynucleotide sequence is used as a method of treating cancer, viral infection or an autoimmune disease. More particularly, the identified polynucleotide encoding a test polypeptide is transduced into antigen presenting cells and the transduced antigen presenting cells are administered directly to a mammal via a parenteral route to treat cancer, a viral infection or an autoimmune disease. Furthermore, the expression vector containing at least the polynucleotide encoding a test polypeptide and a cell binding element is administered directly into a mammal via a parenteral route to treat cancer, a viral infection or an autoimmune disease.

A further embodiment of the present invention is a method of producing a vaccine to immunize a mammal comprising the steps of: transducing antigen presenting cell by introducing the expression vector of the present invention into a cell and expressing said vector to produce an antigen under conditions wherein said antigen is secreted from the cell. In specific embodiments, antigen presenting cells are transduced with the antigen in vitro or ex vivo prior to administering the antigen presenting cells to the mammal. All of the vaccines of the present invention can be administered parenterally.

In specific embodiments, the method of inducing an immune response comprises the step of co-administering to an organism the expression vector and a cytokine expression vector. A number of studies have shown that the responses to individual plasmids can be enhanced by co-administration of a cytokine expressing plasmid. It should be noted that picogram to nanogram quantities of locally synthesized cytokine from the expression vector are too low to have systemic effects on the whole mammal, but can still strongly influence the local cytokine environment and thus the immune response to the administered antigen. Examples of cytokines include, but are not limited to, GM-CSF and IL-2. A skilled artisan readily recognizes that the polynucleotide sequences for a cytokine and the polynucleotide sequences for the antigen can be incorporated into one expression vector; thus eliminating the use of two separate vectors. In addition to cytokines, plasmids that contain unmethylated CpG sequences enhance the cell mediated (Th1) response (Carson et al., 1997). CpG sequence motifs include but are not limited to, RRCpGYY. Thus, a skilled artisan realizes that supplementation of a cytokine with the expression vector or addition of a CpG sequence motif in the present invention would result in the enhancement of the immune response.

In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together; each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple nucleic acid sequences can be efficiently expressed using a single promoter/enhancer to transcribe a single message (U.S. Pat. Nos. 5,925,565 and 5,935,819). Furthermore, a skilled artisan is cognizant that the entire nucleic acid sequence of a gene does not have to be used. Instead, partial nucleic acid sequences of MHC class I and II restricted epitopes can be fused together, resulting in a chimeric fusion gene transcribed by one promoter. For example, a specific embodiment of the present invention is a method of simultaneously inducing both CD4+ and CD8+ T-cells comprising the steps of administering a fusion protein wherein the protein comprises both a MHC-I and MHC-II epitope fused to a cell binding element. Thus, one skilled in the art recognizes that the use of multiple antigenic sequences results in the treatment of a variety of diseases with the administration of one vaccine.

Another specific embodiment of the present invention is a method of inducing an immune response comprising the steps of administering to a mammal one expression vector, wherein said expression vector comprises a polynucleotide sequence encoding a first fusion protein and a polynucleotide sequence encoding a second fusion protein under transcriptional control of one promoter, wherein said first fusion protein comprises a first signal sequence, a first antigen and a first cell binding element and said second fusion protein comprises a second signal sequence, a second antigen and a first cell binding element. In specific embodiments, the first and second signal sequences are the same signal sequence, the first and second antigens are different antigens and the cell binding elements is a Fc fragment. In further embodiments, the first and second signal sequences are the same, the first and second antigens are different antigens and the first and second cell binding elements are the same cell binding elements. Further embodiments include, the first and second signal sequences are different, the first and second antigens are different antigens and the first and second cell binding elements are the same cell binding elements or the first and second signal sequences are the same, the first and second antigens are different antigens and the first and second cell binding elements are different cell binding elements. An additional embodiment includes that the polynucleotide sequence encoding the first fusion protein and the polynucleotide sequence encoding the second fusion protein are under separate-transcriptional control, and wherein the polynucleotide sequence encoding the first fusion protein and the polynucleotide sequence encoding the second fusion protein are in tandem in one expression vector.

One skilled in the art is cognizant that multiple nucleic acid sequences can be cloned into the vector in tandem such that each nucleic acid sequence is a separate entity. Each entity contains a promoter that drives the expression of the individual nucleic acid sequence resulting in expression of separate antigens from one vector. This technique efficiently expresses nucleic acid sequences using multiple promoters to transcribe the individual messages.

A further embodiment of the present invention is a method of producing a fusion protein comprising the steps of introducing the expression vector of the present invention into a cell and expressing said vector to produce a fusion protein under conditions wherein said fusion protein is secreted from the cell. In specific embodiments, antigen presenting cells are transduced with the fusion protein in vitro. More particularly, the fusion protein is administered parenterally to a mammal.

A specific embodiment of the present invention is a method of secreting an intracellular protein comprising the steps of introducing an expression vector into a cell, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an intracellular protein, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and expressing said vector to produce a fusion protein under conditions wherein said fusion protein is secreted from the cell. More specifically, the polynucleotide sequence encoding the intracellular protein is truncated or mutated to increase efficiency of secretion. In specific embodiments, the intracellular protein is HPV 16 E7.

Another specific embodiment of the present invention is a method of secreting a membrane protein comprising the steps of introducing an expression vector into a cell, wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding a membrane protein, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence, all operatively linked and expressing said vector to produce a fusion protein under conditions wherein said fusion protein is secreted from the cell. More specifically, the polynucleotide sequence encoding the membrane protein is truncated or mutated to increase efficiency of secretion. In specific embodiments, the membrane protein is EBV nuclear antigen 1.

The invention also includes a kit comprising the composition of the invention and an instructional material that describes adventitially administering the composition to a cell or a tissue of a mammal. In another embodiment, this kit comprises a (preferably sterile) solvent suitable for dissolving or suspending the composition of the invention prior to administering the compound to the mammal.

Dosage and Formulation

The expression vectors, transduced cells and fusion proteins (active ingredients) of this invention can be formulated and administered to treat a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of the organism. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The active ingredient can be administered orally in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups, emulsions and suspensions. The active ingredient can also be formulated for administration parenterally by injection, rapid infusion, nasopharyngeal absorption or dermoabsorption. The agent may be administered intramuscularly, intravenously, or as a suppository.

Gelatin capsules contain the active ingredient and powdered carriers such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium Ethylenediaminetetraacetic acid (EDTA). In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field.

The active ingredients of the invention may be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs that are described in U.S. Pat. Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and 4,406,890. Other adjuvants, which are useful, include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate and dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL-12. Other components may include a polyoxypropylene-polyoxyethylene block polymer (Pluronic.RTM.), a non-ionic surfactant, and a metabolizable oil such as squalene (U.S. Pat. No. 4,606,918).

Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

Useful pharmaceutical dosage forms for administration of the compounds of this invention can be illustrated as follows.

Capsules: Capsules are prepared by filling standard two-piece hard gelatin capsulates each with 100 milligram of powdered active ingredient, 175 milligrams of lactose, 24 milligrams of talc and 6 milligrams magnesium stearate.

Soft Gelatin Capsules: A mixture of active ingredient in soybean oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules are then washed and dried.

Tablets: Tablets are prepared by conventional procedures so that the dosage unit is 100 milligrams of active ingredient. 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of cornstarch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or to delay absorption.

Injectable: A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredients in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

Suspension: An aqueous suspension is prepared for oral administration so that each 5 milliliters contain 100 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0 grams of sorbitol solution U.S.P. and 0.025 milliliters of vanillin.

Accordingly, the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in an mammal body to achieve a particular effect (see, e.g., Rosenfeld et al., 1991; Rosenfeld et al., 1991 a; Jaffe et al., supra; Berkner, supra). One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

The active ingredients of the present invention can be provided in unit dosage form wherein each dosage unit, e.g., a teaspoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and mammal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical composition in the particular host.

These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

Lipid Formulation and/or Nanocapsules

In certain embodiments, the use of lipid formulations and/or nanocapsules is contemplated for the introduction of the expression vector, into host cells.

Nanocapsules can generally entrap compounds in a stable and/or reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 .mu.m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and/or such particles may be easily made.

In a specific embodiment of the invention, the expression vector may be associated with a lipid. The expression vector associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. The lipid or lipid/expression vector associated compositions of the present invention are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates which are not uniform in either size or shape.

Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which are well known to those of skill in the art which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Phospholipids may be used for preparing the liposomes according to the present invention and may carry a net positive, negative, or neutral charge. Diacetyl phosphate can be employed to confer a negative charge on the liposomes, and stearylamine can be used to confer a positive charge on the liposomes. The liposomes can be made of one or more phospholipids.

A neutrally charged lipid can comprise a lipid with no charge, a substantially uncharged lipid, or a lipid mixture with equal number of positive and negative charges. Suitable phospholipids include phosphatidyl cholines and others that are well known to those of skill in the art.

Lipids suitable for use according to the present invention can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma Chemical Co., dicetyl phosphate ("DCP") is obtained from K & K Laboratories (Plainview, N.Y.); cholesterol ("Chol") is obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20oC. Preferably, chloroform is used as the only solvent since it is more readily evaporated than methanol.

Phospholipids from natural sources, such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine are preferably not used as the primary phosphatide, i.e., constituting 50% or more of the total phosphatide composition, because of the instability and leakiness of the resulting liposomes.

"Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). However, the present invention also encompasses compositions that have different structures in solution than the normal vesicular structure. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and/or the presence of divalent cations. Liposomes can show low permeability to ionic and/or polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and/or results in an increase in permeability to ions, sugars and/or drugs.

Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and/or neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic and/or electrostatic forces, and/or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and/or by transfer of liposomal lipids to cellular and/or subcellular membranes, and/or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one may operate at the same time.

Liposome-mediated oligonucleotide delivery and expression of foreign DNA in vitro has been very successful. Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.

In certain embodiments of the invention, the lipid may be associated with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the lipid may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the lipid may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression vectors have been successfully employed in transfer and expression of an oligonucleotide in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

Liposomes used according to the present invention can be made by different methods. The size of the liposomes varies depending on the method of synthesis. A liposome suspended in an aqueous solution is generally in the shape of a spherical vesicle, having one or more concentric layers of lipid bilayer molecules. Each layer consists of a parallel array of molecules represented by the formula XY, wherein X is a hydrophilic moiety and Y is a hydrophobic moiety. In aqueous suspension, the concentric layers are arranged such that the hydrophilic moieties tend to remain in contact with an aqueous phase and the hydrophobic regions tend to self-associate. For example, when aqueous phases are present both within and without the liposome, the lipid molecules may form a bilayer, known as a lamella, of the arrangement XY-YX. Aggregates of lipids may form when the hydrophilic and hydrophobic parts of more than one lipid molecule become associated with each other. The size and shape of these aggregates will depend upon many different variables, such as the nature of the solvent and the presence of other compounds in the solution.

Liposomes within the scope of the present invention can be prepared in accordance with known laboratory techniques. In one preferred embodiment, liposomes are prepared by mixing liposomal lipids, in a solvent in a container, e.g., a glass, pear-shaped flask. The container should have a volume ten-times greater than the volume of the expected suspension of liposomes. Using a rotary evaporator, the solvent is removed at approximately 40oC. under negative pressure. The solvent normally is removed within about 5 min. to 2 hours, depending on the desired volume of the liposomes. The composition can be dried further in a desiccator under vacuum. The dried lipids generally are discarded after about 1 week because of a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipid in sterile, pyrogen-free water by shaking until all the lipid film is resuspended. The aqueous liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed under vacuum.

In the alternative, liposomes can be prepared in accordance with other known laboratory procedures: the method of Bangham et al. (1965), the contents of which are incorporated herein by reference; the method of Gregoriadis, as described in DRUG CARRIERS IN BIOLOGY AND MEDICINE, G. Gregoriadis ed. (1979) pp. 287-341, the contents of which are incorporated herein by reference; the method of Deamer and Uster, 1983, the contents of which are incorporated by reference; and the reverse-phase evaporation method as described by Szoka and Papahadjopoulos, 1978. The aforementioned methods differ in their respective abilities to entrap aqueous material and their respective aqueous space-to-lipid ratios.

The dried lipids or lyophilized liposomes prepared as described above may be dehydrated and reconstituted in a solution of inhibitory peptide and diluted to an appropriate concentration with an suitable solvent, e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer. Unencapsulated nucleic acid is removed by centrifugation at 29,000xg and the liposomal pellets washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50-200 mM. The amount of nucleic acid encapsulated can be determined in accordance with standard methods. After determination of the amount of nucleic acid encapsulated in the liposome preparation, the liposomes may be diluted to appropriate concentrations and stored at 4oC. until use.

A pharmaceutical composition comprising the liposomes will usually include a sterile, pharmaceutically acceptable carrier or diluent, such as water or saline solution.

Gene Therapy Administration

One skilled in the art recognizes that the expression vector of the present invention can be utilized for gene therapy. For gene therapy, a skilled artisan would be cognizant that the vector to be utilized must contain the gene of interest operatively linked to a promoter. For antisense gene therapy, the antisense sequence of the gene of interest would be operatively linked to a promoter. One skilled in the art recognizes that in certain instances other sequences such as a 3' UTR regulatory sequences are useful in expressing the gene of interest. Where appropriate, the gene therapy vectors can be formulated into preparations in solid, semisolid, liquid or gaseous forms in the ways known in the art for their respective route of administration. Means known in the art can be utilized to prevent release and absorption of the composition until it reaches the target organ or to ensure timed-release of the composition. A pharmaceutically acceptable form should be employed which does not ineffectuate the compositions of the present invention. In pharmaceutical dosage forms, the compositions can be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. A sufficient amount of vector containing the therapeutic nucleic acid sequence must be administered to provide a pharmacologically effective dose of the gene product.

One skilled in the art recognizes that different methods of delivery may be utilized to administer a vector into a cell. Examples include: (1) methods utilizing physical means, such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein said vector is complexed to another entity, such as a liposome, aggregated protein or transporter molecule.

Accordingly, the present invention provides a method of transferring a therapeutic gene to a host, which comprises administering the vector of the present invention, preferably as part of a composition, using any of the aforementioned routes of administration or alternative routes known to those skilled in the art and appropriate for a particular application. Effective gene transfer of a vector to a host cell in accordance with the present invention to a host cell can be monitored in terms of a therapeutic effect (e.g. alleviation of some symptom associated with the particular disease being treated) or, further, by evidence of the transferred gene or expression of the gene within the host (e.g., using the polymerase chain reaction in conjunction with sequencing, Northern or Southern hybridizations, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection, mRNA or protein half-life studies, or particularized assays to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted in level or function due to such transfer).

These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

Furthermore, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts can vary in in vitro applications depending on the particular cell line utilized (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector employed for gene transfer to replicate in that cell line). Furthermore, the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the vector, as well as also the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis). One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.

It is possible that cells containing the therapeutic gene may also contain a suicide gene (i.e., a gene which encodes a product that can be used to destroy the cell, such as herpes simplex virus thymidine kinase). In many gene therapy situations, it is desirable to be able to express a gene for therapeutic purposes in a host cell but also to have the capacity to destroy the host cell once the therapy is completed, becomes uncontrollable, or does not lead to a predictable or desirable result. Thus, expression of the therapeutic gene in a host cell can be driven by a promoter although the product of said suicide gene remains harmless in the absence of a prodrug. Once the therapy is complete or no longer desired or needed, administration of a prodrug causes the suicide gene product to become lethal to the cell. Examples of suicide gene/prodrug combinations which may be used are Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.

Claim 1 of 21 Claims

We claim:

1. An expression vector comprising a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked, wherein said polynucleotide encoding an antigen and said polynucleotide encoding a cell binding element are interchangeably linked.




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